NDLI: Mechanisms of DNA Demethylation in Chicken Embryos PURIFICATION AND PROPERTIES OF A 5-METHYLCYTOSINE-DNA GLYCOSYLASE

Content Provider	Journal of Biological Chemistry (JBC)
Author	Jost, Jean-Pierre Siegmann, Michel Sun, Lijie Leung, Roland
Abstract	We have previously shown that in developing chicken embryos and differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs through the replacement of 5-methylcytosine by cytosine (Jost, J. P.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4685-4688; Jost, J. P. & Jost, Y. C. (1994) J. Biol. Chem. 269, 10040-10043). We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from 12-day-old chicken embryos. The enzyme copurifies with a mismatch-specific thymine-DNA glycosylase and an apyrimidic-endonuclease. The reaction product of the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified apyrimidic-endonuclease activity cleaves 3′ from the apyrimidic sugar. A 52.5-kDa peptide, isolated as a single band from preparative SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase activities. 5-Methylcytosine-DNA glycosylase has an apparent pI of 5.5-7.5 and maximal activity between pH 6.5 and 7.5. The K for hemimethylated oligonucleotide substrate is 8 × 10M with a Vmax of 4 × 10 mol/h/μg protein. 5-Methylcytosine-DNA glycosylase binds equally well to methylated and non-methylated DNA. The enzyme reacts six times faster with the hemimethylated DNA than with the same bifilarly methylated DNA sequence, and single-stranded methylated DNA is not a substrate. The action of the enzyme is distributive.
Related Links	http://www.jbc.org/content/270/17/9734.abstract
Ending Page	9739
Starting Page	9734
Page Count	6
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	17
Volume Number	270
DOI	10.1074/jbc.270.17.9734
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1995-04-28
Access Restriction	Open
Subject Keyword	Nucleic Acids, Protein Synthesis, and Molecular Genetics
Alternative Title	Mechanisms of DNA Demethylation in Chicken Embryos
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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